E. coli RNA polymerase (holoenzyme) forms rifampicin-resistant pre- initiation complexes at specific sites in Adenovirus 2 DNA (Surzycki, Armstrong, and Gussin, unpub. data); this observation provides a way of investigating two types of questions: (1) Are these sites those at which eucaryotic RNA polymerase initiates transcription? 2) Are there rifampicin-resistant pre-initiation sites in nuclear DNA or in integrated adenovirus DNA in transformed cells? Answers to these questions would provide insight into the temporal and spatial control of Ad 2 gene expression during productive infection and into possible differences in initiation sites for viral and chromosomal transcription. Pre-requisite to these studies is the quantification of the rifampicin-resistant pre-initition sites in Ad 2 DNA, first by measuring the incorporation of gamma-p32-ATP or -GTP into RNA by rifampicin- resistant complexes, and second by comparing the amount of enzyme needed to saturate Ad 2 DNA with the amount needed to saturate a DNA template of known transcription specificity (e.g., phage lambda DNA). The relationship of RNA made in vitro to the three classes of in vivo RNA (early, late, and transformed) can be studied by competition hybridization or by electron microscopic visualization of RNA-DNA hybrids. Topographical identification of transcription units can be facilitated by EM denaturation mapping of Ad 2 DNA and DNA fragments made by shearing or by enzymatic degradation by bacterial restriction enzymes. The specificity of eucaryotic and coli RNA polymerases can be compared further by competition for binding sites on Ad 2 DNA. To answer the second kind of question posed above, we will examine the formation of pre-initiation complexes of coli RNA polymerase with nuclear DNA from uninfected (permissive and non-permissive) cells as well as from cells transformed by Ad 2.